Peptide

ABSTRACT

The present invention provides peptides which affect T-cells, presumably by action on the T-cell antigen receptor. The present invention further relates to the therapy of various inflammatory and autoimmune disease states involving the use of these peptides. Specifically, the peptides are useful in the treatment of disorders where T-cells are involved or recruited. The peptide is of the following formula: A-B-C-D-E in which: A is absent or 1 or 2 hydrophobic amino acids, B is a positively charged amino acid, C is a peptide consisting of 3 to 5 hydrophobic amino acids, D is a positively charged amino acid, and E is absent or up to 8 hydrophobic amino acids.

This application is a 371 of PCT/AU96/0018 filed Jan. 16, 1996.

The present invention relates to peptides which affect T-cells,presumably by action on the T-cell antigen receptor. The presentinvention further relates to the therapy of various inflammatory andautoimmune disease states involving the use of these peptides.Specifically, the peptides are useful in the treatment of disorderswhere T-cells are involved or recruited.

T-cells are a subgroup of cells which together with other immune celltypes (polymorphonuclear, eosinophils, basophils, mast cells, B-, NKcells), constitute the cellular component of the immune system. Underphysiological conditions T-cells function in immune surveillance and inthe elimination of foreign antigen(s). However, under pathologicalconditions there is compelling evidence that T-cells play a major rolein the causation and propagation of disease. In these disorders,breakdown of T-cell immunological tolerance, either central orperipheral, is a fundamental process in the causation of autoimmunedisease.

Central tolerance involves thymic deletion of self reactive cells(negative selection) and positive selection of T-cells with low affinityfor self major histocompatibility complex antigens (MHC). In contrast,there are four, non-mutually exclusive hypotheses that have beenproposed to explain peripheral T-cell tolerance which are involved inthe prevention of tissue specific autoimmune disease. These include:anergy (loss of co-stimulatory signals, down regulation of receptorscritical for T-cell activation), deletion of reactive T-cells, ignoranceof the antigen by the immune system and suppression of autoreactiveT-cells. Tolerance once induced does not necessarily persistindefinitely. A breakdown in any of these mechanisms may lead toautoimmune disease.

Autoimmune disease and other T-cell mediated disorders are characterisedby the recruitment of T-cells to sites of inflammation. T-cells at thesesites, coupled with their ability to produce and regulate cytokines andinfluence B-cell function, orchestrate the immune response and shape thefinal clinical outcome. An understanding of the process of antigenrecognition and subsequent T-cell activation, leading to T-cellproliferation and differentiation, is therefore pivotal to both healthand disease. The critical component of antigen recognition on thesurface of T-cells is the complex antigen receptor (TCR) which is amultisubunit structure that recognises antigen in the context ofMHC-encoded proteins on the surface of antigen-presenting cells.Disturbance in this intricate structure-function relationship of theTCR, integrating antigen recognition with T-cell activation may providethe therapeutic means to deal with inflammation and T-cell mediateddisorders.

The TCR is composed of at least seven transmembrane proteins. Thedisulfide-linked (αβ-Ti) heterodimer forms the clonotypic antigenrecognition unit, while the invariant chains of CD3, consisting of ε, γ,δ, and ζ and η chains, are responsible for coupling the ligand bindingto signalling pathways that result in T-cell activation and theelaboration of the cellular immune responses. Despite the gene diversityof the TCR chains, two structural features are common to all knownsubunits. Firstly, they are transmembrane proteins with a singletransmembrane spanning domain--presumably alpha-helical. Secondly, allthe TCR chains have the unusual feature of possessing a charged aminoacid within the predicted transmembrane domain. The invariant chainshave a single negative charge, conserved between the mouse and human,and the variant chains possess one (TCR-β) or two (TCR-α) positivecharges. Listed below in TABLE 1 is the transmembrane sequence of TCR-αin a number of species showing that phylogenetically this region ishighly conserved indicating an important functional role. Thesubstitutions between species are very conservative.

                  TABLE 1                                                         ______________________________________                                        Sequence comparison of TCR-α transmembrane region                       SPECIES SEQUENCE                                                              ______________________________________                                        MOUSE   NLSVMGLRILLLKVAGFNLLMTL (SEQ ID NO: 1)                                RAT          NLSVMGLRILLLKVAGFNLLMTL (SEQ ID NO: 1)                           SHEEP          NLSVTVFRILLLKVVGFNLLMTL (SEQ ID NO: 2)                         COW          NLSVI VFRILLLKVVGFNLLMTL (SEQ ID NO: 3)                          HUMAN      NLSVI GFRILLLKVAGFNLLMTL (SEQ ID NO: 4)                            ______________________________________                                    

Studies on the assembly of the multicomponent TCR by Manolios et al(1990, 1991, 1994) showed that the stable interaction between TCR-α andCD3-δ and TCR-α and CD3-ε was localised to eight amino acids within thetransmembrane domain of TCR-α (shown above in bold) and it was thecharged amino acids arginine and lysine that were critical for thisprocess. This finding exemplified the fact that amino acids within thetransmembrane domain not only functioned to anchor proteins but wereimportant in the assembly of subunit complexes and protein-proteininteractions.

The above system depended on the modification of complementary strandDNA (cDNA) to create a number of protein mutants. Chimeric cDNAmolecules were transfected into COS (fibroblast line) cells to expressthe required protein. Coexpression of these chimeric proteins were usedto evaluate the region of interaction. Reiterating the above, thetechnology involved cDNA manipulation, metabolic labelling,immunoprecipitation and gel electrophoresis.

Transmembrane domains are small in size and proteins transversing thisregion are usually constrained to an alpha-helical configuration. Thesebiophysical features coupled with the ability to engineerprotein-protein interactions via transmembrane charge groups suggestedto the present inventor a possible new approach to intervene andpotentially disturb TCR function.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the delayed induction and clinical severity of disease inanimals treated or untreated with the core peptide.

FIG. 1B shows the average weight of animals treated or untreated withthe core peptide.

The present inventor has developed a series of peptides that areinhibitors of function of this crucial receptor, presumably byinterfering with assembly. The present inventor has also found thatthese peptides have an effect on T-cell mediated inflammation and thatcarboxyl terminal conjugation did not alter the function of thepeptides. This is exemplified by coupling peptide to a lipid carriersystem with increased effect and no loss of function. In addition, thepresent inventor has also found that the peptide alone had the abilityto translocate intracellularly making it a potentially effective drugdelivery system. The efficacious clinical manifestations of theadministered peptide would be a decrease in inflammation, e.g. asdemonstrated by a decrease of arthritis in an adjuvant model ofarthritis.

Accordingly, in a first aspect the present invention consists in apeptide of the following formula:

A-B-C-D-E in which:

A is absent or 1 or 2 hydrophobic amino acids

B is a positively charged amino acid

C is a peptide consisting of 3 to 5 hydrophobic amino acids

D is a positively charged amino acid, and

E is absent or up to 8 hydrophobic amino acids

In a preferred embodiment of the present invention C is 3 or 4hydrophobic amino acids.

In a further preferred embodiment of the present invention A is 2hydrophobic amino acids and E is 1 to 3, and preferably 1, hydrophobicamino acids.

In yet a further embodiment of the present invention B is arginine and Dis lysine or B is lysine and D is arginine.

In yet a further preferred embodiment of the present invention thepeptide is Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val (SEQ ID NO: 5),Leu-Lys-Ile-Leu-Leu-Leu-Arg-Val (SEQ ID NO: 6),Gly-Phe-Arg-Ile-Leu-Leu-Leu-Lys-Val (SEQ ID NO: 7) orPhe-Lys-Ile-Leu-Leu-Leu-Arg-Val (SEQ ID NO: 8).

In a second aspect the present invention consists in a therapeuticcomposition comprising the peptide of the first aspect of the presentinvention and a pharmaceutically acceptable carrier.

In a third aspect the present invention consists in a method of treatinga subject suffering from a disorder in which T-cells are involved orrecruited, the method comprising administering to the subject atherapeutically effective amount of the composition of the second aspectof the present invention.

The therapeutic composition may be administered by any appropriate routeas will be recognised by those skilled in the art Such routes includeoral, transdermal, intranasal, parenteral, intraarticular andintraocular.

In a fourth aspect the present invention consists in a method ofdelivering a chemical moiety to a cell comprising exposing the cell tothe chemical moiety conjugated to the peptide, preferably to the carboxyterminal, as claimed in any one of claims 1 to 10.

A non-exhaustive list of disorders in which T-cells areinvolved/recruited include:

Allergic diathesis e.g. delayed type hypersensitivity, contactdermatitis

Autoimmune disease e.g. systemic lupus erythematosus, rheumatoidarthritis, multiple sclerosis, diabetes, Guillain-Barre syndrome,

Hashimotos disease, pernicious anemia

Gastroenterological conditions e.g. Inflammatory bowel disease, Crohn'sdisease, primary biliary cirrhosis, chronic active hepatitis

Skin problems e.g. psoriasis, pemphigus vulgaris

Infective disease e.g. AIDS virus, herpes simplex/zoster

Respiratory conditions e.g. allergic alveolitis,

Cardiovascular problems e.g. autoimmune pericarditis

Organ transplantation

Inflammatory conditions e.g. myositis, ankylosing spondylitis.

As used herein the term "subject" is intended to cover both human andnon-human animals.

As will be recognised from the above discussion the peptide of thepresent invention is based on a portion of transmembrane domain ofTCR-α. The complete murine sequence of this portion isNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 9), whereas the correspondinghuman sequence is NLSVIGFRILLLKVAGFNLLMTL (SEQ ID NO: 4). There iscomplete sequence homology across a range of species in the last 15amino acids of the TCR-alpha chain distal to the sequence upon thepeptide of the present invention is based (shown in bold). Peptidesincluding these additional 15 residues may have activity similar to thepeptide of the present invention. The essential feature is that thepeptide includes two positively charged amino acids separated by 3 to 5hydrophobic amino acids. Further, as will be clear from the followingexamples, the peptide of the present invention may be modified at thecarboxy terminal without loss of activity. Accordingly, it is intendedthat the present invention includes within its scope peptides whichinclude additional amino acids to the "core" sequence of the peptide ofthe present invention and which affect the T-cell antigen receptor.

As demonstrated in the following examples the peptide of the presentinvention is able to enter cells. Accordingly it is envisaged that,apart from its other uses, the peptide of the present invention could beused as a "carrier" to deliver other therapeutic agents to cells. Thiscould be achieved, for example, by conjugating the therapeutic to bedelivered into the cell to the peptide of the present invention.

As will be readily understood by those slklled in this field hydrophobicamino acids are Ala, Val, Leu, Ile, Pro, Phe, Tyr and Met, whilstpositively charged amino acids are Lys, Arg and His.

In order that the nature of the present invention may be more clearlyunderstood, preferred forms thereof will now be described with referenceto the following examples.

EXAMPLE 1 Synthesis of Peptide

The first step was to synthesise a short hydrophobic peptidecorresponding to the predetermined assembly sequence. The amino acidsequence of the competitive peptide is NH₂-Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val-OH (SEQ ID NO: 5) hereafterreferred to as "core peptide". Subsequently a number of other peptideslisted in TABLE 2 were synthesised (>95% purity, by Auspep Australia,Melbourne, Australia) and examined for their effect on T-cell functionand inflammation.

                                      TABLE 2                                     __________________________________________________________________________    Peptides and their sequence                                                   PEPTIDE                                                                            SEQUENCE                                                                 __________________________________________________________________________          Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val-OH (SEQ ID NO: 5)                   peptide                                                                       A                Met-Gly-Leu-Arg-IIe-Leu- Leu-Leu-OH (SEQ ID NO: 10)          B                Leu-Gly-Ile-Leu-Leu-Leu-Gly-Val-OH (SEQ ID NO: 17)           C                      Leu-Lys-IIe-Leu-Leu-Leu-Arg-Val-OH (SEQ ID NO: 6)      D                Leu-Asp-Ile-Leu-Leu-Leu-Glu-Val-OH (SEQ ID NO: 12)           E                Leu-Arg-Ile-Leu-Leu-Leu-Ile-Lys-Val-OH (SEQ ID NO: 13)       F                Leu-Arg-Leu-Leu-Leu-Lys-Val-OH (SEQ ID NO:                   __________________________________________________________________________         14)                                                                  

EXAMPLE 2 Solubility

The core peptide and other peptides listed above were noted to behydrophobic and insoluble in aqueous solutions. A variety of solventsand carriers were tested. These included ethanol, dimethylsulphoxide(DMSO), dimethyl formamide (DMF), trifluoracetic acid (TFA), squalaneoil (2,6,10,15,19,23-hexamethyltetracosane), and lipid conjugation byaddition of palmitic acid to the core peptide via TRIS-conjugation(Whittaker R.G., Bender V. J. 1991) to increase solubility. Thepreferred solvent was DMSO and the final concentration used in cellcultures ranged from 0.1%-0.2%. Concentrations of DMSO greater than 1%was toxic to cells. Stock solutions of peptide and lipopeptideconjugates were dissolved in DMSO and used in a 1/1000 dilution.

The addition of peptide/lipopeptide in DMSO to aqueous solutionsresulted in "fat" or "crystal" globules that settled to the bottom ofthe tissue culture flask and dissolved poorly. These globules could beseen by phase contrast microscopy, but were less obvious for lipidconjugates.

Core peptide containing C¹⁴ -glycine (C¹⁴ -peptide) was synthesised byAuspep Australia and used to study solubility. C¹⁴ -peptidedissolved/suspended in DMSO was added to a final concentration of 100 μMto T-cell media (RPMI 1640 supplemented with 10% foetal calf serum and0.3% mercaptoethanol: TCM) and shaken. The media was centrifuged andsupernatant filtered through 0.2 μM filter or left unseparated. Thetotal radioactivity in unseparated medium was 20.000 cpm, 1000 cpm afterthe medium was centrifuged and 500 cpm after the media was filtered.These experiments highlight the insoluble nature of the peptide in vitroand suggest that approximately 5% goes into solution.

EXAMPLE 3 Entry of Peptide into Cells

To examine if peptide enters cells, C¹⁴ -peptide was added to a flask of5×10⁶ 2B4.11 cells (T-cell hybridoma specific for cytochrome c) in afinal concentration of 100 μM and 0.2% DMSO and incubated overnight. Theadherent cells were washed four times with phosphate buffered saline(PBS) in the flask, solubilised with triton-containing buffer andradioactivity counted. The amount of radioactivity in the supernatantseas 70,000 cpm and 5000 cpm in the solubilised cells.

In a variation of the above experiment, 2B4.11 cells (7.5×10⁴) weregrown in Petri dishes containing 2 ml of TCM and a "Transwell" with 0.4μM membrane was placed in the Petri dish. C¹⁴ -peptide in a finalconcentration of 100 μM and 0.1% DMSO were added in the "Transwell" andafter 24 hr and 48 hr incubation the counts determined on both sides ofthe filter and in the cells. Approximately 85% of radioactivity wasretained in the "Transwell", 8% in the Petri dish media and 7% withincells. The above experiments demonstrated that peptide was able to entercells. Considering the low solubility of peptide (5%-10%) all of theavailable peptide in solution entered the cells (7%).

EXAMPLE 4 Intracellular Localisation of Fluoresceinated Peptide inT-cells

Experiments suggested that the small percentage of peptide that goesinto solution can enter/or be taken up by cells. To confirm this, corepeptide covalently linked with fluorescein isothiocyanate (FITC) wasadded to T-cells and intracellular localisation determined byvisualisation using confocal or conventional UV light microscopy.

Fluoresceinated labelled core peptide was prepared as follows: 10.25 mgof core peptide was dissolved in 0.5 ml dimethylformamide (DMF) and 2 μMof FITC in 0.5 ml of DMF was added dropwise with stirring, at roomtemperature. The pH was adjusted to 9 with N-methyl,N,N-diisopropylamine, and the reaction allowed to proceed for 1 hr.Semi-preparative HPLC was then used to separate FITC-peptide from freeFITC, using a C-4 column (6 ml/min; buffer A, 0.1% TFA; buffer B, 80%acetonitrile, 20% water; 0.1% TFA; linear gradient of 40%-100% B).Fractions were monitored by analytical HPLC and the fractions containingpure fluoresceinated core peptide (FITC-peptide) pooled.

Two flasks of cultured 2B4.11 cells (5×10⁶) were spun down andresuspended in PBS containing calcium and magnesium. To one flask, FITCdissolved in DMSO was added to a final concentration of 10 μM and to theother FITC-peptide 10 μM. The final concentration of DMSO in both flaskswas 0.1% previously shown to have no effects on T-cells. The cells wereincubated at 37° C. for 30 min and then examined under the confocalmicroscope.

The observations can be summarised as follows: (i) FITC and FITC-peptideentered the cells; (ii) free HITC gave brighter fluorescence thanFITC-peptide in the cells; (iii) the intracellular staining pattern wasnot different between the free FITC and FITC-peptide. Nuclear andespecially bright nucleolar staining was observed; (iv) conjugation ofpeptide by FITC did not prevent entry of peptide into cells; (v) therewas no "leaching" out of cells of FITC-peptide over a 5 hr period. Theseexperiments demonstrate that FITC-peptide could be taken up by cells andlocalised intracellularly. In conjuction with experiments previouslydescribed showing intracellular uptake by C¹⁴ -peptide it is evidentthat it is the inherent nature of the peptide sequence and not itsconjugates (FITC, C¹⁴) that allows cellular entry.

EXAMPLE 5 Tris-fat Conjugation of Core Peptide Carboxyl Terminal

The effect of carboxyl conjugation of core peptide, as exemplified bylipid conjugation, on the ability of peptide to competitively inhibitthe function of this crucial receptor was investigated. The efficaciousclinical manifestations of the administered lipopeptide would be adecrease in inflammation e.g. as demonstrated by a decrease of arthritisin an adjuvant model of arthritis, as would be seen with peptide. Inaddition to the lipoconjugation of core peptide a number of otherlipopeptides were synthesised and used as controls in subsequentexperiments. The lipopeptides were synthesized according to the methodsset out in Whittaker, R. G., Hayes, P. J., and Bender, V. J. (1993)Peptide Research 6, 125 and Australian Patent No. 649242. The disclosureof these references is incorporated by herein by cross reference

Preparation of Fluorescein LabelledGly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val-Gly-Tris-mono- and tri-palmitates.To a solution of each of the deprotected lipopeptides (15 and 6 mg) inDCM (1 ml) a solution of FITC (4 mg 10 μmole) in DMF (500 μl) was addedwith stirring. The apparent pH of the reaction was maintained at 9.0 bythe addition of triethyl amine (TEA). The fluorescein-labelled mono, andtri-palmitoyl derivative of the peptide were purified bysemi-preparative HPLC (C4 column, System B). The purified compounds wereevaporated to diyness and lyophilised from tert. butyalcohol to give thefluorescein labelled peptide monopalmitate (R_(t) ^(B), 7.83) andtripalmitate (R_(t) ^(B) 9.85) which were tested as described below.

TLC of the fluorescein-labelled lipopeptides (DCM: MeOH, 95:5) showedthe absence of free FITC and free Gly-Tris-monoparnitate andGly-Tris-tripalmitate (used in lipopeptide synthesis) (by ninhydrinstaining).

Solid Phase Peptide Synthesis. Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val (SEQID NO: 5) (core peptide) and its fully protected form,Boc-Gly-Leu-Arg(PMC)-Ile-Leu-Leu-Leu-Lys(Boc)-Val-OH (SEQ ID NO: 16)(and the C14 -labelled peptide) were supplied by Auspep Pty Ltd. Bothwere synthesised by the FMOC-chemistry in the manual mode.

It will also be readily understood by those skilled in the art thatthere are a number of well known linkers that can be used to joincompounds (such as peptides) with a carboxyl group to an amino group.These include:

a) a linker with an amino group to the compound and a carboxyl group tothe Tris (or amino acid if present) such as an amino acid or antibiotic.

b) a linker with an amino group to the compound and a sulphonic acidgroup to the Tris (or amino acid if present) such as2-aminoethanesulphonic acid (taurine).

c) a linker with an hydroxyl group to the compound and a carboxyl groupto the Tris (or amino acid if present) such as glycolic acid, lacticacid etc.

d) a linker with an hydroxyl group to the compound and a sulphonic acidgroup to the Tris (or amino acid if present) such as2-hydroxyethanesulphonic acid (isethonic acid).

e) a linker with an hydroxyl group to the compound and a reactive halidegroup to the Tris (or amino acid if present) such as 2-chloroethanol.

f) other examples of potentially suitable linkers between a compoundwith a reactive carboxyl and the amino group of Tris (or amino acid ifpresent) include the compound families exemplified byp-hydroxybenzaldehyde, 2-chloroacetic acid, 1,2- dibromoethane andethyleneoxide.

Linkers could also contain disulphide groups that would reduce toliberate modified peptide intracellularly.

EXAMPLE 6 Localisation of FITC Conjugated Lipopeptides in COS Cells

Using con-focal microscopy, the ability of FITC-conjugated lipopeptidesto enter non-T cells (COS cells-fibroblasts) was examined.

Materials:

Stock concentration in DMSO--Core peptide.Tris.monopalmitate. FITC (MW1862) 10 mM; core peptide.Tris.dipalmitate.FITC (MW 2334) 10 mM; corepeptide.Tris.tripalmitate.FITC (MW 2806) 10 mM; glycine.Tris.monopalmitate.FITC (MW 805) 10 mM; glycine.Tris.tripalmitate.FITC (MW1286) 10 mM; FITC, (MW 390) 6.4 mM.

Method:

COS cells were grown on coverslips until 80% confluent, washed twicewith PBS and incubated with FITC conjugated lipopeptides for 15 min or 2hr. The final concentration of lipopeptides was 10 μM and 6.4 μM forFITC, for each time point respectively. Cells were washed twice withPBS, mounted with PBS/glycerol and examined with confocal microscopy.

Results:

Experiments indicated that fluorescein-conjugated lipopeptides cantransmigrate across cell membranes and localise to within the cellularcytoplasm, reaching as far as the endoplasmic reticulum (ER), whereprotein synthesis takes place. The extent of cellular penetration wasinfluenced by the lipid moiety attached to the peptide. Of thelipopeptides the monopaletate had the greater ability to infiltratewithin the fibroblasts and T-cells so far examined (see below). The ERis the best site to try and effect assembly. Once all the chains haveassembled and transported to the cell surface it may be much harder todisrupt the receptor at the cell surface membrane. Targetting peptidesto the ER is an ideal site to disrupt the TCR complex.

EXAMPLE 7 Localisation of FITC Conjugated Lipopeptides in T-cells

Using con-focal microscopy, the ability of FITC conjugated lipopeptidesto enter T-cells was examined.

Materials:

Lipopeptides as above. 2B4.11 T-cell hybridoma cell line.

Method:

2B4.11 T-cells were grown in TCM and resuspended in a concentration of8×10⁵ cells/ml. Viability >95% using trypan blue. One ml of cells wasadded to polypropylene tubes and washed twice with PBS. Cells wereresuspended in PBS and one microliter of stock FITC-conjugatedlipopeptides added for 30 min. Cells were washed with PBS, mounted withPBS/glycerol, and viewed using confocal microscopy.

Results:

Similar to that of COS cells (see above). Results showed thatlipopeptides were able to enter T-cells. The lipoconjugation of peptidedoes not prevent entry of peptides into cells and has the potential useof being used as a carrier vehicle to increase solubility.

EXAMPLE 8 Effect of Peptides and Lipopeptides on TCR Assembly and CellSurface Expression on T-cells using Flow Cytometry Analysis

Materials

The T-cell hybridoma 2B4.11 which expresses a complete TCR on the cellsurface was used as a positive control to assess the effects of peptideson TCR expression. The cells were grown in TCM. The β-deficient variant21.2.2 and the β- and ζ-deficient cell line 3.12.29, derived byrepetitive subcloning of 2B4.11 cells (Sussman et al., 1988) and lackingTCR expression were used as negative controls.

Peptides tested included core peptide, lipopeptides and a peptide fromtumor necrosis factor receptor termed 558 (used as a negative control).The final concentration of each substance used in incubation was 10 μM.

Antibodies

The following antibodies were used for immuno-precipitation and flowcytometry analysis: Mouse IgG2a monoclonal antibody (MAb) against TCR-αchain of the T-cell hybridoma 2B4 (A2B4-2, Samelson et al., 1983), MAbagainst 2B4.11 TCR-β chain (K)25), hamster IgG anti-CD3-ε MAb (145-2C11[2C11], Leo et al., 1987), rabbit anti-CD3-ε polyclonal antiserum raisedagainst purified mouse CD3-ε (127, Minami et al., 1987), anti-CD3-δpolyclonal antibody (R9) raised in goat immunized with a COOH-terminalpeptide of the mouse CD3-δ chain (Samelson et al., 1986).

Method

FACS analysis: 1×10⁶ (2B4.11, 21.2.2) cells were incubated with a numberof separate peptides and lipopeptides in a final concentration of 10 μMovernight. The cells were then wvashed with PBS and incubated with 50 μlprimary antibody (A2B4 or 2C11) for 30 mins at 4° C. Cells were washedtwice in PBS and 0.1% BSA and incubated for an additional 30 min at 4°C. with FITC-labelled second antibody. Cells were washed two additionaltimes with PBS and 0.1% BSA prior to analysis on a Becton-Dickson FACSAnalyser or Becton-Dickson FACS Scan.

Results

The expression of TCR on 2B4.11 cells treated with core peptide controlpeptide, or lipopeptides did not alter the cell surface expression ofthe receptor. These experiments have been repeated with higherconcentration of core peptide (100 μM) and longer incubation timesranging from 1-10 days and the results have been the same showing nochange in T-cell surface antigen receptor expression.

The following experiments were performed to assess the in vitro effectsof peptides/lipopeptides on T-cell function.

EXAMPLE 9 Antigen Presentation Assay I

An antigen presentation assay (described below) examined the ability ofa number of peptides to inhibit T-cell activation following antigenrecognition, by measuring the product of T-cell activation,Interleukin-2 (IL-2).

Material. The following cell lines were used: 2B4.11, a T-cell hybridomathat expresses a complete antigen receptor on the cell surface (Samelsonet al., 1983) and produces IL-2 following antigen recognition(cytochrome c). Interleukin-2 dependent T-cell line (CTLL) forconventional biological IL-2 assays; and the B-cell hybridoma cell lineLK 35.2 (LK, I-E^(k) bearing; Kappler et al., 1982) which acts as theantigen presenting cell. The hybridomas were grown in TCM. Cytochrome c(Sigma, USA) was added in the media to give a final concentration of 50μM in the antigen presenting assay.

Peptides examined included: core peptide, seven other control peptidesfrom a variety of sources having an equivalent length to core peptideand peptides A, B, C, D, E and F. The final concentration of thepeptides in the antigen presentation assay was examined at severallevels ranging from 10 μM to 100 μM.

Method. For T-cell antigen stimulation 2×10⁴ LK35.2 cells wereco-cultured with 50 μM pigeon cytochrome c dissolved in PBS and 2×10⁴2B4.11. T-cells for 16 hr. The assay was done in triplicate.Supernatants were recovered and IL-2 content determined by CTLLproliferation. The incorporation of ³ H-thymidine is directlyproportional to the amount of IL-2 present in the supernatant. Theability of different peptides to inhibit IL-2 production was examined.In addition to measuring ³ H-thymidine incorporation, IL-2 measurements(IU/ml) were also determined.

Results. In assays where either cytochrome c (antigen) or LK cells(antigen presenting cells) were omitted there was no IL-2 production.The lack of T-cell activation under such conditions indicated that therewas no lipopolysaccharide (LPS) or endotoxin in the solutions which mavhave non-specifically stimulated the T-cells. The combination of allthree constituents of the assay at the concentrations shown aboveresulted in the production of IL-2 as measured by high ³ H-thymidineincorporation by CTLL cells (22,000 cpm). When core peptide or otheranalogues were added to the assay system the amount of IL-2 producedvaried respectively. All peptides tested at 10 μM had no effect on IL-2production. The best effect was noted with core peptide at 100 μM andpeptide C (100 μM) leading to a 15%-30% reduction in IL-2 productioncompared to control. This was reproducible on at least three separateoccasions. Peptides A, B, D, E and F had a variable and minor effect onT-cell activation. The seven control peptides with equivalent length butno sequence homology to the peptide had no effect on IL-2 production.

Preincubation of core peptide and other peptides at 37° C. in TCM priorto addition in the antigen presenting assay, improved solubility andactivity and was reflected as an additional incremental increase abovebaseline activity noted with freshly prepared peptide.

EXAMPLE 10 Antigen Presentation Assay II

The following experiments were performed to assess the in vitro effectsof lipopeptides on T-cell function.

Material. Lipopeptides examined included: core peptideTris-monopalmitate (100 μM and 0.1% DMSO) and core peptideTris-tripalmitate (100 μM and 0.2% DMSO). The final concentration of thetwo lipopeptides in the antigen stimulation assay is shown in bracketsrespectively.

Method. As described in Example 9.

Results. Initially, when core peptide Tris-tripalmitate (100 μM and 0.2%DMSO) was added, there was a reduction in T-cell activation by 75%(highest count 5.190 cpm cf 22,000 cpm for control). The addition ofcore peptide Tris-monopalmitate (100 μM and 0.1% DMSO) had a profoundeffect on IL-2 production, with only 137 cpm recorded (similar tobackground). The concentration of 0.1% DMSO and 0.2% DMSO used in thetest system was examined and not found to influence IL-2 production.Subsequent experiments have confirmed these findings and show an IL-2reduction of 86%-92% compared to control. Palmitic acid alone (100 μM)used to conjugate the peptide, added to the antigen presenting assaysystem did not affect IL-2 production.

The following experiments examined the ability of core peptide tocirculate within the animal following administration, and the effects onexperimentally induced inflammation.

EXAMPLE 11 Distribution of C¹⁴ -core Peptide

To examine the distribution of subcutaneously injected peptide in mice,C¹⁴ -core peptide (5 mg/mouse) was dissolved in 150 microliters ofsqualane oil and injected at the base of the tail of Balbic mice. After24 hr, counts were measured from pulped organs. Distribution of thepeptide was noted in thymus (5%), spleen (7%), blood (3%) and a largeproportion in lymph nodes (28%), kidney (30%) and liver (28%).

Experiments were extended to examine the ability of core peptide toprevent disease in animal models of inflammation. Three in vivoexperimental models including adjuvant induced arthritis in rats,cyclophosphamide induced diabetes in NOD mice, and experimental allergicneuritis in rats were used. In these models, encompassing two species,core peptide was able to influence the degree of inflammation.

EXAMPLE 12(a) Adjuvant Induced Arthritis in Rats

The rat adjuvant arthritis model is a classic model of inflammationwhich has been used extensively by a number of laboratories to studydisease progression and effects of potential new anti-inflaimatory drugsthereon over the last 30 years (Pearson et al., 1961; Cremer et al.,1990; Holmdahl and Kvick., 1992; Cannon et al.,1993). This model hasalso been widely used by researchers at the Royal North Shore Hospitalover the last 10 years and procedures have been established for thestudy of this model of inflammation. All procedures on the animals werecarried out under halothane/oxygen/nitrous oxide anaesthesia (2% v/vhalothane in 1 liter/min O₂ and 2 liters/minN₂ O). Rats were injectedintradermally at the base of the tail with a minimal adjuvant dose (1 mgheat killed Mycobacterium tuberculosis [MTB9 in 100 μl squalane) onceand only once. The method for coadmainistrating test samples with MTBwas first described by Whitehouse et al., 1990. At regular intervalsbetween days 0-28, animals were weighed and their arthritic conditionassessed by measurement of maximum tail thickness and rear paw thickness(with a micrometer screw gauge). Rats were housed in holding bins afterthe initial tail injection and allowed access to unlimited water andpellet food. On day 29 the animals were sacrificed.

Materials. The first experiment consisted of 12 rats weighingapproximately 190-210 grams that were purchased from the Perth AnimalResource Centre (ARC) and maintained in the Gore Hill Animal Housefacility. Used were core peptide (30 mg) suspended in adjuvant (0.6 mlsqualane containing 7 mg MTB), core peptide Tris-monopalmitate (15 mg)suspended in 0.6 ml adjuvant, core peptide Tris-tripalmitate 20 mg/0.6ml of adjuvant.

Rats were divided into four groups, each group containing three rats.First group received adjuvant only (positive control), second groupadjuvant with core peptide, third group core peptide.Tris. monopalmitatesuspended in adjuvant, and last group core peptide.Tris. tripalmitate inadjuvant. Rats were injected with the above compounds in a 0.1 ml volumeat the base of the tail. Baseline measurements of rat weight, paw width,and tail diameter were made on Day 0, and subsequently on day 4, 7, 9,14, 16, 18, 21, 25 and 28. Arthritis was graded and animals sacrificedif there was marked swelling, redness and obvious discomfort. Not allrats given MTB developed arthritis. In general more than 80% of controlrats developed arthritis.

Results. After 18 days all the control animals given adjuvant only haddeveloped arthritis and had to be sacrificed. Two of the three corepeptide treated animals (2/3) had no evidence of arthritis. Similarly,two of the three animals given core peptide.Tris.tripalmnitate had noevidence of arthritis. Animals given core peptide.Tris.monopalmitate andadjuvant all developed arthritis. However, the onset and development ofarthritis in this latter group was prolonged by 3-4 days and theclinical severity was much reduced (number of joints, paw swelling, lossof weight) compared to controls.

Experiments using adjuvant induced arthritis in rats showed that thepeptide and its lipid conjugate had a protective effect on the inductionof arthritis in this animal model. Results of repeat and subsequentexperiments using a number of different peptides (7 mg/rat) and drugsare summarised in TABLE 3.

                  TABLE 3                                                         ______________________________________                                        Effects of different peptides on adjuvant induced arthritis in rats.                            INDUCTION OF ARTHRITIS                                      PEPTIDE   MTB ALONE   WITH PEPTIDE EFFECT                                     ______________________________________                                           CORE   3/3 (100%)  1/3 (33%)    Protective                                                              1/5 (20%)                                                                                  Protective                                                5/5 (100%)                                                                          1/4 (25%)                                                                                   Protective                          A                   2/4 (50%)                                                                              4/6 (67%)                                                                               Protective                             B                   2/4 (50%)                                                                              2/4 (50%)                                                                                   No effect                          C                   4/5 (80%)                                                                              0/4 (0%)                                                                                     Protective                        D                   4/5 (80%)                                                                              4/5 (80%)                                                                                   No effect                          E                   5/5 (100%)                                                                            3/5 (60%)                                                                                    Protective                         F                   5/5 (100%)                                                                            0/5 (0%)                                                                                      Protective                        CS*               5/5 (100%)                                                                              1/5 (20%)                                                                                    Protective                         DXM*             5/5 (100%)                                                                               4/4 (100%)                                                                                 No effect+                           ______________________________________                                         CS*, cyclosporin, 50 mg/kg; DXM, dexamethasone (2 mg/kg).                     +, animals developed arthritis but the onset of arthritis was delayed by      3-4 days.                                                                

The results of the above experiments indicated that core peptide had aneffect on inflammation both to delay its onset, decrease severity, andprevent onset of disease. These effects were similar to those obtainedwith the co-administration of cyclosporin and adjuvant. Cyclosporin is awell known and widely used immunosuppressive agent. There was noindiscriminate effect of peptide action. Best results were noted withcore peptide, peptide C and F. Core peptide and peptide C each havecharged amino acid groups at the same site but the amino acids reversed.This indicated that it was the charge group rather than the particularamino acid that was important. In contrast there was no effect notedwith peptide B or D having either no or negative charge group aminoacids respectively. Extending the amino acids downstream towards thecarboxy terminus had no negative effect. This observation confirms thatcarboxy modification can be performed without loss of biologicalactivity. Therefore these peptides can be used as carrier peptides forthe delivery of other chemical moieties. Increasing the amino acidnumber between the two polar charge groups (peptide E) resulted in lowerefficacy. Decreasing the number of amino acids between the chargedgroups (peptide F) had no negative effect.

EXAMPLE 12(b) Dosage Effect of Core Peptide

Based on previous experiments by Whitehouse et al (personalcommunication) an initial dose of 5-7 mg/rat was given. To evaluate alower limit, a number of different core peptide concentrations wereexamined. The results (TABLE 4) indicated that in addition to a specificaction of core peptide it was limited by its effects by dosage.

                  TABLE 4                                                         ______________________________________                                        Effect of core peptide dosage on adjuvant induced arthritis.                  INDUCTION OF ARTHRITIS                                                        PEPTIDE                                                                              MTB ALONE  WITH PEPTIDE                                                                              EFFECT OF PEPTIDE                               ______________________________________                                        7    mg    11/13 (85%)                                                                              3/12 (25%)                                                                              Protective                                    3.5 mg        4/5 (80%)                                                                                 2/5 (40%)                                                                              Protective                                 1.7 mg         6/7 86%)                                                                                   7/8 (88%)                                                                             No effect                                 ______________________________________                                    

EXAMPLE 12(c) Tail Diameter Measurements

A feature of adjuvant induced arthritis is the development ofinflammation in the tail. Tail measurements (mm) between saline injectedrats and core peptide treated rats were not statistically significant.Tail diameters from MTB treated rats however were significantlyincreased (p<0.001) compared to saline and core peptide treated rats(p<0.001), TABLE

                  TABLE 5                                                         ______________________________________                                        Effect of core peptide on tail inflammation as assessed by tail                thickness (mm)                                                                                        DAY                                                      TREATMENT                                                                            0      5       10    15    20    25                                ______________________________________                                        SALINE (n = 4)                                                                           7.13   7.75    7.95  8.48  8.78  8.80                              MTB only (n = 5)                                                                              6.86                                                                              8.80   8.96  9.46 9.60   9.73                             MTB + PEPTIDE                                                                                     7.62   8.24  8.76 8.96   9.10                             (n = 5)                                                                       ______________________________________                                    

In addition to the oedema noted in the tails, MTB alone given to ratscaused ulceration and inflammation at the site of injection that was notpresent with rats given core peptide or saline.

EXAMPLE 13 Experimental Allergic Neuritis (EAN)

To further confirm the ability of core peptide to delay and diminish theseverity of disease induced by T-cells a different model (experimentalallergic neuritis) was tested "blind" by independent experimenters(Associate Professor Pollard and Mr J Taylor) at a different institution(University of Sydney).

Materials and Methods

Animals. Male Lewis rats weighing between 239-451 grams were obtainedfrom the Bosch Animal House, University of Sydney colony or from ARC,Perth. All experiments were conducted in accordance with experimentalguidelines approved by the Animal Care and Ethics Committee of theUniversity of Sydney.

Induction of EAN. Lewis rats were immunised in each hind footpad with50-75 μl of bovine peripheral nerve myelin (PNM) emulsified in completeFreunds adjuvant. The immunisation emulsion consisted of equal volumesof saline and incomplete Freunds adjuvant (Sigma, USA) mixed with bovinePNM and MTB (strain H37RA, DIFCO) added at 15 mg/ml and 5 mg/mlrespectively. Where animals received ovalbumin/peptide the peptide wasadded to the imnmnisation emulsion at 70 mg/ml. These experiments wereperformed with the experimenters having no knowledge of what peptide wasin the immunisation emulsion.

Animals were observed at least every second day post immunisation forclinical signs and were scored using the following scale: 0, normal;0.5, weak tail; 1, flaccid tail; 1.5, limp tail and ataxia in hind legs;2, paraparesis; 2.5, limp tail and severe paraparesis; 3, paraplegia;3.5, limp tail and paraplegia and forelimb paresis; 4, quadraparesis;4.5 limp tail and quadraplegia; 5, dead.

Peripheral Nerve Myelin Isolation. Bovine PNM was prepared essentiallyas described by Norton and Podulso (1973).

Results.

A representative example is shown in FIG. 1. In this experiment corepeptide delayed induction and clinical severity of disease. Similar datawere observed for peptide C. These data confirm the efficacy of corepeptide and peptide C as general immunosuppressants.

EXAMPLE 14 Diabetes in NOD/Lt (F) Mice

In yet another model of T-cell mediated disease the effects ofsubcutaneously injected core peptide on the induction of diabetes inNOD/Lt (F) mice was tested "blind" by an independent experimenter (ProfL Harrison, WEHI, Melbourne).

A cellular autoimmune process that selectively destroys the pancreaticislet beta cells is thought to be responsible for the development ofinsulin-dependent diabetes mellitus (IDDM) in humans and in thespontaneous animal models including the NOD mouse (Leiter et al., 1987).A common histopathological feature associated with the development ofIDDM is insulitis, the presence within and around the islets ofmononuclear cells consisting predominantly of T lymphocytes and to alesser extent macrophages (Foulis et al., 1986). Experimental strategiesaimed at suppressing cellular autoimmunity such as neonatal thymectomy,administration of cyclosporin A or administration of anti-T lymphocyteantibodies prevent the development of diabetes (Campbell et al., 1991).

Animals

NOD/Lt(F) mice 10 weeks old at experimental Day 0. This is ahigh-incidence strain commonly used as an animal model for diabetes.

Materials

Core peptide dissolved in squalane at a stock concentration of 3.33mg/ml. Ovalbumin used as a control was suspended in squalane at a stockconcentration of 3.33 mg/ml. Peptide and ovalbumin were "solubilised"just prior to injection, with vortexing. A total of 250 μg (75 μl) wasinjected subcutaneously on the right flank on Day -1, 0 and 1. Mice weregiven intraperitoneal cyclophosphamide in water at 300 mg/kg on Day 0.Blood glucose measurements were taken on Day 0, 10, 14 and 21. Therewere 16 mice in the treatment group and 16 in the ovalbumin controlgroup.

Results. Experiments demonstrate a protective effect of core peptide onthe induction of autoim-mune beta cell destruction which manifests asdiabetes (TABLE 6). This finding again confirms the generalimmunosuppressive ability of core peptide in a different T-cell mediateddisease model.

                  TABLE 6                                                         ______________________________________                                        Effects of core peptide on the induction of diabetes in NOD/Lt (F) mice              PERCENTAGE OF MICE DEVELOPING DIABETES                                 TREATMENT                                                                              DAY 0     DAY 10    DAY 14  DAY 21                                   ______________________________________                                        Ovalbumin                                                                              0%        0%        42%     65%                                      (n = 16)                                                                      Peptide       0%        0%           5%                                                                                  12%                                (n = 16)                                                                      ______________________________________                                    

Summary

In recent years a vast number of different methods have been used anddevised to interfere with the interaction between TCR, MHC or antigen(trimolecular complex) and thereby influence immune responses. Thetherapeutic potential associated with the development of these ideas andmethods of application has not been overlooked. The strategies haveincluded the use of monoclonal antibodies to block MHC or TCRinteractions, bloclkng antibodies to important co-stimulatory orregulatory proteins on the T-cell surface, vaccination with diseaseinducing T-cells, or TCR epitopes, competing antigens, and inhibition ofcytokines or their receptors. The ability to disrupt TCR function by aspecific competitive peptide designed to affect assembly has not beenpreviously reported and examined. The present inventor has clearly shownthat the peptides of the present invention are able to inhibit T-cellmediated immune responses in at least three different models by amechanism previously unreported.

The experiments described herein were principally conducted with a corepeptide deliberately chosen to be homologous with the known sequence ofthe mouse and rat TCR alpha chain. However, for clinical treatment ofhumans a peptide containing a phenylalanine residue instead of leucinetowards the amino terminus of the core peptide may be preferable tomaximise homology with the known human sequence (TABLE 1).

It will be appreciated by persons sldlled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

Bansal et al. (1993). Curr Ther Res 54:1

Bevins C. L., Zasloff M. (1990). Am Rev Biochem. 59, 395

Campbell, I. L., Kay, T., Oxbrow, L., Harrison, L. (1991).J.Clin.Invest87, 739.

Cannon et al. (1993) Arthritis and Rheum. 36:126

Clevers H., Alarcon B., Wileman T., Terhorst C. (1988). Am Rev. Immunol.6,629

Cosson P., Lankford S. P., Bonifacino J. S., Klausner R., (1991). Nature351, 414

Cremer et al. (1990). Clin Exp Immunol 82:307

DeGrado W. F., Musso G. F., Lieber M., Kaiser E. T., Kezdy F. J. (1982).Biophys J. 37, 329

Foulis, A., Liddle, C. N., Farquharson, M., Richmond, J. (1986).Diabetologia. 29, 267.

Francis et al. (1989). Arthritis and Rheum. 32:608.

Hart et al. (1993). Eur. J. Immunol. 23:1588

Hashida et al. (1991). Agents Actions 34:242.

Higashijima T., Uzu S., Nakajima T., Ross E. M. (1988). J. Biol Chem263, 6491

Holmdahl R and Kvick. (1992). Clin Exp Immunol 88:96

James J. A. and Harley J. B. (1992). J. Immunol. 148, 1074

Kappler J., White J., Wegmann E., Mustain E., Marrack P. (1982) PNAS79,3604

Leiter, E., Prochaska M., Coleman, D. (1987). Amer. J. Pathol. 128,380-393.

Manolios N., Bonifacino J. S., Klausner R. D., (1990) Science, 248, 274

Manolios N., Letourner F., Bonifacino J. S., Klausner R. D. (1991) EMBOJ. 10, 1643

Manolios N. (1992). Todays Life Sciences 4,32

Manolios N., Kemp, O., Li. ZG (1994). Eur. J. Immunol. 24-89

Pearson et al. (1961). J Exp. Med 113.485

Samelson L. E., Germain R. N., Schwartz R. W., (1983). Proc NatlAcad.Sci U.S.A. 80,6971

Takagi et al. (1987). Pathology. 19:294

Whitehouse et al. (1990). Agents actions. 31:47

Whittaker, R. G., Bender V. J. (1991). Proceedings of the SecondInternational Conference on Solid Phase Synthesis and RelatedTechnologies, Canterbury, UK, August 1991 (Patent Entitled "Amino Acids,Peptides or Derivatives Thereof Coupled to Fats" filed in AustraliaDecember 1990. PCT/AU90/00599).

Whittaker, R. G., Hayes, P. J., and Bender, V. J. (1993) PeptideResearch 6, 125 and Australian Patent No. 649242.

Zhang et al. (1990). J. Immunol 145:2489

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 16                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #Leu Leu Lys Val Ala Glyly Leu Arg Ile Leu                                    #                 15                                                          -  Phe Asn Leu Leu Met Thr Leu                                                             20                                                               - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #Leu Leu Lys Val Val Glyal Phe Arg Ile Leu                                    #                 15                                                          -  Phe Asn Leu Leu Met Thr Leu                                                             20                                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #Leu Leu Lys Val Val Glyal Phe Arg Ile Leu                                    #                 15                                                          -  Phe Asn Leu Leu Met Thr Leu                                                             20                                                               - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #Leu Leu Lys Val Ala Glyly Phe Arg Ile Leu                                    #                 15                                                          -  Phe Asn Leu Leu Met Thr Leu                                                             20                                                               - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 -  Gly Leu Arg Ile Leu Leu Leu Lys Val                                          1               5                                                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 -  Leu Lys Ile Leu Leu Leu Arg Val                                              1               5                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 -  Gly Phe Arg Ile Leu Leu Leu Lys Val                                          1               5                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 -  Phe Lys Ile Leu Leu Leu Arg Val                                              1               5                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 28 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #Leu Leu Lys Val Ala Glyly Leu Arg Ile Leu                                    #                 15                                                          #Ser Sersn Leu Leu Met Thr Leu Arg Leu Trp                                    #             25                                                              - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                -  Met Gly Leu Arg Ile Leu Leu Leu                                              1               5                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                -  Leu Gly Ile Leu Leu Leu Gly Val                                              1               5                                                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                -  Leu Asp Ile Leu Leu Leu Glu Val                                              1               5                                                           - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                -  Leu Arg Ile Leu Leu Leu Ile Lys Val                                          1               5                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                -  Leu Arg Leu Leu Leu Lys Val                                                  1               5                                                           - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                -  Gly Leu Arg Ile Leu Leu Leu Lys Val Gly                                    #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                     #Gly = "Boc-Gly"HER INFORMATION:                                                        (A) NAME/KEY: Other                                                           (B) LOCATION: 3                                                     #Arg = "Arg[PMC]"ER INFORMATION:                                                        (A) NAME/KEY: Other                                                           (B) LOCATION: 8                                                     #Lys = "Lys[Boc]"ER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                -  Gly Leu Arg Ile Leu Leu Leu Lys Val                                          1               5                                                           __________________________________________________________________________

I claim:
 1. A therapeutic composition comprising a pharmaceuticallyacceptable carrier and a peptide of the following formula:A-B-C-D-E inwhich:A is absent, or glycine and 1 hydrophobic amino acid, or 1 or 2hydrophobic amino acids B is a positively charged amino acid C is apeptide consisting of 4 hydrophobic amino acids D is a positivelycharged amino acid, and E is 1 to 8 hydrophobic amino acids, wherein thepeptide is not ARLPVLKLV, RVMAPRALL or VKLFPVKLFP.
 2. A therapeuticcomposition as claimed in claim 1 in which A is 2 hydrophobic aminoacids.
 3. A therapeutic composition as claimed in claim 1 in which E is1 to 3 hydrophobic amino acids.
 4. A therapeutic composition as claimedin claim 3 in which E is 1 hydrophobic amino acid.
 5. A therapeuticcomposition as claimed in claim 1 in which B is arginine and D is lysineor B is lysine and D is arginine.
 6. A method of treating a subjectsuffering from a disorder in which T-cells are involved or recruited,the method comprising administering to the subject a therapeuticallyeffective amount of the therapeutic composition as claimed in claim 1.7. A peptide in which the peptide is Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val(SEQ ID NO:5).
 8. A peptide in which the peptide isGly-Phe-Arg-Ile-Leu-Leu-Leu-Lys-Val (SEQ ID NO:7).
 9. A peptide in whichthe peptide is Leu-Lys-lle-Leu-Leu-Leu-Arg-Val (SEQ ID NO:6).
 10. Apeptide in which the peptide is Phe-Lys-Ile-Leu-Leu-Leu-Arg-Val (SEQ IDNO:8).
 11. A method of inhibiting T-cell function in a subject,comprising administering to the subject an amount effective to inhibitT-cell function of a peptide of the following formula:A-B-C-D-E inwhich:A is absent, or glycine and 1 hydrophobic amino acid, or 1 or 2hydrophobic amino acids B is a positively charged amino acid C is apeptide consisting of 4 hydrophobic amino acids D is a positivelycharged amino acid, and E is absent or up to 8 hydrophobic amino acids.12. A method of delivering a chemical moiety to a cell, comprisingexposing the cell to the chemical moiety conjugated to a peptide of thefollowing formula:A-B-C-D-E in which:A is absent, or glycine and 1hydrophobic amino acid, or 1 or 2 hydrophobic amino acids B is apositively charged amino acid C is a peptide consisting of 4 hydrophobicamino acids D is a positively charged amino acid, and E is absent or upto 8 hydrophobic amino acids.